Process for treating polysaccharides produced by fermentation

ABSTRACT

IN THE TREATMENT OF POLYSACCHARIDES OBTAINED BY FERMENTATION IN A MUST OR BROTH AND FROM WHICH THE POLYSACCHARIDE IS RECOVERED BY PRECIPITATION, THE IMPROVEMENT WHICH COMPRISES HEATING THE BROTH PRIOR TO PRECIPITATION AT A TEMPERATURE WITHIN RANGE OF 80* TO 130*C. FOR A TIME WITHIN THE RANGE OF 10 TO 120 MINUTES WHILE AT A PH WITHIN THE RANGE OF 6.3 TO 6.9.

United States Patent 3,591,578 PROCESS FOR TREATING POLYSACCHARIDES PRODUCED BY FERMENTATION Pierre Colin and Victor Guibert, Melle, Deux-Sevres, France, assignors to Melle-Bezons, Melle, Deux-Sevres, France No Drawing. Filed Sept. 6, 1968, Ser. No. 758,102 Claims priority, application France, Apr. 29, 1968, 484 Int. Cl. C07c 47/18 US. Cl. 260-209 6 Claims ABSTRACT OF THE DISCLOSURE In the tretament of polysaccharides obtained by fermentation in a must or broth and from which the polysaccharide is recovered by precipitation, the improvement which comprises heating the broth prior to precipitation at a temperature within the range of 80 to 130 C. for a time within the range of 10 to 120 minutes while at a pH within the range of 6.3 to 6.9.

The present invention relates to a process for the treatment of polysaccharides produced by fermentation.

Polysaccharide gums produced through glucide fermentation by microorganisms, especially microorganisms of the type Xanthomonas, such as Xanthomonas campestris, are heteropolysaccharides in which the principal compo nents are glucose, mannose and glucuronic acid. These heteropolysaccharides may contain acetyl groups and another component thereof may be pyruvic acid. The physical properties of these gums in the rheologic field are very special. The viscosity of their colloidal dispersions or sols is very stable in the presence of salts or inorganic or organic acids.

The pseudo-plastic character of these sols demands that their viscosity be defined under well established conditions. For all viscosity measurements referred to herein, use was made of a Brookfield viscosimeter LVT, at speeds of 30 rpm, needle No. 3.

US. Pat. No. 3,355,447 teaches that it is possible to avoid degradation in time of sols of polysaccharides produced by fermenttaion and separation through precipitation by the introduction of an alcohol into the fermented must or broth if the must or broth has previously been subjected to heating to a temperature within the range of 60 to 78 C. for about 20 minutes. In accordance with the teachings of the patent, heating to a temperature above 78 C. is undesirable.

It has now been surprisingly found that by carrying out the heating step of the fermented must or broth without previous dilution, at a pH within the range of 6.3 to 6.9 and Within a temperature range of 80 to 130 C., for about 10 to 120 minutes, not only is the degradation in time of the polysaccharide subsequently isolated from the must or broth by precipitation avoided, but in addition their rheologic characteristics are improved, especially in their acid or salt-containing aqueous sols. In the preferred practice of this invention, the temperature for the heating step is about 95 C. with the pH below 6.8 and heating for about one-half hour. For example, at 85 C. the heating time may advantageously Patented July 6, 1971 ice be 2 hours or more whereas, at 120 C. a heating time of 10 minutes is sufficient.

For precipitating the polysaccharide from the fermented must 0r broth previously heated in accordance with the process of this invention, there are introduced one or more diluents which are effective to cause polysaccharide precipitation, such for example as an alcohol such as methanol, ethanol, isopropanol or tertiary butanol, or acetone or mixtures thereof. In carrying out the present process, it has been found to be especially advantageous to make use of tertiary butanol which avoids the necessity of using, additionally, a solution of an electrolyte such as potassium or sodium chloride. On the other hand, such an electrolyte is necessary to provide an economical precipitation operation when methanol or ethanol is employed as the precipitating diluent. Preferably, the amount of tertiary butanol used is Within the range of 0.7 to 1.3 volumes per volume of fermented must or broth. Corresponding volumes of the other diluents may be used.

The following examples are given by way of illustration, but not by way of limitation, of the practice of this invention:

EXAMPLE 1 In a fermentation vat, 50 liters of a must formulated of an aqueous solution of saccharose having a concentration of 20 grams per liter, soya-bean oil cake (4 g. per 1.), disodium hydrogen phosphate having 12 mols of water of crystallization (10 g. per 1.) and magesium sulfate having 7 mols of water of crystallization (0.25 g. per 1.) are fermented by the microorganism Xanthomonas campestris with continuous stirring and aeration. After 64 hours fermentation, the sugar has been consumed and the must contains 15 g. per liter of polysaccharides in the form of a sol. The must has a viscosity of 4500 centipoises (cps.) and a pH of 6.5 such that it is unnecessary to adjust its pH before treatment in accordance with the practice of this invention.

By injecting steam into the vat, the must is raised to a temperature of C. within 10 minutes and is maintained at this temperature for one-half hour. The contents of the vat are then transferred within 10 minutes to a vessel having a capacity of 150 liters and provided with a stirring device and a cooling jacket through which water is circulated at a temperature of 14 C. When the vat has been emptied, the temperature of the must in the vessel is 40 C.

60 liters of tertiary butanol are added to the vessel to cause polysaccharide precipitation. After standing for 30 minutes, liters of supernatant liquid are withdrawn and 10 liters of tertiary butanol are poured onto the precipitate in the vessel. After 15 minutes standing, 10 liters of liquid are withdrawn and 10 liters of fresh tertiary butanol are poured on to the precipitate and then the bulk of the precipitate and liquid is subjected to filtration through a wire gauze. There is obtained 4.7 kg. of a fibrous precipitate which is dried for one hour at a temperature of 98 C. 720 g. of dry polysaccharide is finally obtained and ground.

For purposes of comparison, a check-precipitate is prepared in the same manner without previous heating of the fermented must in accordance with the practice of this invention.

Starting with the two precipitates, two series of sols are prepared containing 1% by weight polysaccharides in the following diluents:

AWater B-Aqueous solution of NaCl at by weight GAqueous solution of NaCl at 26% by Weight DAqueous solution of KCl at by weight E-Aqueous solution of MgSO at 5% by weight F-Aqueous solution of NaCl (8% by weight), CaCl (2.5% by weight) GAqueous solution of HCl at by weight TABLE II Heating tempera tlge) of the must HAqueous solution of H PO at 15% by weight Heamngume Ofthemust 85 95 105 115 125 IAqueous solution of HOOC-CH at 60% by weight gg 5; a c1 rid e: 15802c1g0 The viscosity of the various sols is measured 24 hours 15 g8 51% :000 after preparation. The results (in cps.) are given in the 120minutes "N111: .II 2530 2:240 21120 2104011111: following Table I.

TABLE I A B c D E F G H I Check-must (without previous heating) 1, 640 1,580 1, 540 1,600 1,620 1, 580 600 1,400 1, 600 Previously heated must. 2,200 2,400 2,000 2, 480 2, 000 2, 000 2,000 2,200 2,480

It will be seen that there is a significant difference be- 25 TABLE III tween the two series of sols, especially with respect to the viscosity figure in the presence of HCl. Heating z g the must EXAMPLE 2 Heating time of the must 85 95 105 115 125 The viscosity of polysaccharide sols having a concen- None Check-polysaccharid 600 cps tration of 1% by weight in the various diluents of Example g8 h5g8 938 $28 1,620 l was measured. To compare the results with one another, 60 minutes. II 2:300 1:800 1:780 1:480 III: use is made, on the one hand, of a check-precipitate of 2,000 1,120 1,200 1,000 polysaccharides obtained by precipitation without previous heating of the fermented must and, on the other hand, precipitates obtained after previous heating of the TABLE IV same fermented must at various temperatures and for H t t various times. The must was produced by fermentation mg fio g must of a saccharose solution with Xanthomonas compestris and Heating time of the must 85 95 it had a pH of 6.8 so that it was not necessary to adjust 40 None C the pH before treatment in accordance w1th the present 2y 86 00 86 03??? -6 540 055 inventl 30 es 2,000 1,980 1,920 1,200 1, 740 The check-precipitate was obtained as follows: 20 g. per 3 n fig l gig fl gg ggg 8 liter of potassium chloride was added to the fermented must and then 0.7 volume of tertiary butanol per volume r of must was introduced for precipitation. The polysaccharide reci itate was se arated b filtration throu h a fine g g g and then y g y treatment ivith th The1 resulils sect1 forth in the foregoing tables verify that tertiary butanol and dried for one hour at a temperature e PO an processed. In accordance Wlth the of 98 C- and then ground. tree of this 1I1V6I1I1OIl, WhI1 1n the form of aqueous sols, To obtain the other precipitates in accordance with g heated to reltmely hlgh temperatures. Wlthout any the practice of this invention, the operations were carried 3 t ted detrimental consequences wlt.h rfaspect to out in a similar way but the musts were previously heated e r Ogle properties. of the S015 and alsqwlth Improve to temperatures indicated in Tables II, III and IV and mints m 511.011 P mp "F compared W1 th h check' were maintained at the selected temperatures for the g i t faspeclauy m the Y f S015 hydro times indicated. The heating steps were carried out in an c on.C.ac1d mefilum and also sols m Vanous acld Salt autoclave by introduction of live steam. The autoclave was provided with a jacket through which cold water was .2 d e apparent. from the E that We have circulated to accelerate cooling after the heating step. g w and Improved method the treatment The viscosity measurements were carried out with O poysacchandes Produced by.ferment.ano.n Wlth mlcro' samples of 300 g. of sols containing 3 g. of polysacofgainsms P K havmg LmProvFd charides. The sols reach stable viscosity figures after a o oglcdcharactenstlcs and resistance to degradatlon are time of 1 to 2 hours. However, to avoid variations resultb d ing from higher or lower swelling quickness, the viscosid t 1 f 2 f that changes be made m F ties (cps) in the tables below were obtained 24 hours e m S 0 9 atlon.and qperatlon .wlthout depamrig after preparation of the S01. 60 from the spirit of the invention, especially as defined in Table II gives the average figures of the determinations f f clalms effected on the polysaccharide sols in aqueous solutions 1 having the following formulations: n a proqess for lmproylpg i vlscoslty of polysac' charides obtained by precipitation from a fermented sodfum at 5% y Welght 70 must or broth in which they have been produced by fersodlunfl chloflde'at 26% y g mentation, the improvement wherein the fermented must Potassium chloride at 10% by weight or broth, prior to precipitation, and without previous dilu- Magnesium sulfate at 5% by weight tion is heated to a temperature within the range of NaCl (8%)+CaCl (2.5% by weight) to 130 C. for a time within the range of 10 to min- Acetic acid at 60% by weight 75 utes at a pH within the ranges of 6.3 to 6.9.

2. A process as claimed in claim 1 in which the must References Cited or broth is at a pH within the range of 6.3 to 6.8. UNITED STATES PATENTS 3. A process as claimed in claim 1 in which the must or broth is heated for about one-half hour. 10/1956 0pm et a1 260-209 2,818,357 12/1957 Ziegler et al 260 -209 4. A process as clalmed in claim 1 m which the must 5 3 094 517 6/1963 Stanley 260F209 or broth is heated to a temperature of about 95 C. for 3:146:200 8/1964 gg 26O 209 about one-half hour.

5. A process as claimed in claim 1 in which the must ELBE-RT L, ROBERTS, Primary Examiner or broth is at a pH within the range of 6.3 to 6.8. J R

6. A process as claimed in claim 1 in which the must 1O BROWN Asslstant Exammer or broth is heated to a temperature of about 95 C. for U5, (:1, X3, about one-half hour at a pH between 6.3 to 6.8. 195-28 

